Use of noggin, an alk5 inhibitor and a protein kinase c activator, to produce endocrine cells

ABSTRACT

The present invention provides methods to promote the differentiation of pluripotent stem cells into insulin producing cells. In particular, the present invention provides a method to produce cells expressing markers characteristic of the pancreatic endocrine lineage that co-express NKX6.1 and insulin and minimal amounts of glucagon.

This is a continuation of U.S. patent application Ser. No. 15/457,956,filed Mar. 13, 2017, which is a continuation of U.S. patent applicationSer. No. 14/840,297, filed on Aug. 31, 2015, issued as U.S. Pat. No.9,593,310, which is a continuation of U.S. patent application Ser. No.12/970,330, filed on Dec. 16, 2010, issued as U.S. Pat. No. 9,150,833,which claims the benefit of U.S. Provisional Application No. 61/289,671,filed on Dec. 23, 2009. All of the prior applications are incorporatedby reference herein in their entirety.

FIELD OF THE INVENTION

The present invention provides methods to promote the differentiation ofpluripotent stem cells into insulin producing cells. In particular, thepresent invention provides a method to produce cells expressing markerscharacteristic of the pancreatic endocrine lineage that co-expressNKX6.1 and insulin and minimal amounts of glucagon.

BACKGROUND

Advances in cell-replacement therapy for Type I diabetes mellitus and ashortage of transplantable islets of Langerhans have focused interest ondeveloping sources of insulin-producing cells, or β cells, appropriatefor engraftment. One approach is the generation of functional β cellsfrom pluripotent stem cells, such as, for example, embryonic stem cells.

In vertebrate embryonic development, a pluripotent cell gives rise to agroup of cells comprising three germ layers (ectoderm, mesoderm, andendoderm) in a process known as gastrulation. Tissues such as, forexample, thyroid, thymus, pancreas, gut, and liver, will develop fromthe endoderm, via an intermediate stage. The intermediate stage in thisprocess is the formation of definitive endoderm. Definitive endodermcells express a number of markers, such as, HNF3 beta, GATA4, MIXL1,CXCR4 and SOX17.

Formation of the pancreas arises from the differentiation of definitiveendoderm into pancreatic endoderm. Cells of the pancreatic endodermexpress the pancreatic-duodenal homeobox gene, PDX1. In the absence ofPDX1, the pancreas fails to develop beyond the formation of ventral anddorsal buds. Thus, PDX1 expression marks a critical step in pancreaticorganogenesis. The mature pancreas contains, among other cell types,exocrine tissue and endocrine tissue. Exocrine and endocrine tissuesarise from the differentiation of pancreatic endoderm.

Cells bearing the features of islet cells have reportedly been derivedfrom embryonic cells of the mouse. For example, Lumelsky et al. (Science292:1389, 2001) report differentiation of mouse embryonic stem cells toinsulin-secreting structures similar to pancreatic islets. Soria et al.(Diabetes 49:157, 2000) report that insulin-secreting cells derived frommouse embryonic stem cells normalize glycemia in streptozotocin-induceddiabetic mice.

In one example, Hori et al. (PNAS 99: 16105, 2002) disclose thattreatment of mouse embryonic stem cells with inhibitors ofphosphoinositide 3-kinase (LY294002) produced cells that resembled βcells.

In another example, Blyszczuk et al. (PNAS 100:998, 2003) reports thegeneration of insulin-producing cells from mouse embryonic stem cellsconstitutively expressing Pax4.

Micallef et al. reports that retinoic acid can regulate the commitmentof embryonic stem cells to form PDX1 positive pancreatic endoderm.Retinoic acid is most effective at inducing Pdx1 expression when addedto cultures at day 4 of embryonic stem cell differentiation during aperiod corresponding to the end of gastrulation in the embryo (Diabetes54:301, 2005).

Miyazaki et al. reports a mouse embryonic stem cell line over-expressingPdx1. Their results show that exogenous Pdx1 expression clearly enhancedthe expression of insulin, somatostatin, glucokinase, neurogenin3, p48,Pax6, and Hnf6 genes in the resulting differentiated cells (Diabetes 53:1030, 2004).

Skoudy et al. reports that activin A (a member of the TGF-β superfamily)upregulates the expression of exocrine pancreatic genes (p48 andamylase) and endocrine genes (Pdx1, insulin, and glucagon) in mouseembryonic stem cells. The maximal effect was observed using 1 nM activinA. They also observed that the expression level of insulin and Pdx1 mRNAwas not affected by retinoic acid; however, 3 nM FGF7 treatment resultedin an increased level of the transcript for Pdx1 (Biochem. J. 379: 749,2004).

Shiraki et al. studied the effects of growth factors that specificallyenhance differentiation of embryonic stem cells into PDX1 positivecells. They observed that TGF-β2 reproducibly yielded a higherproportion of PDX1 positive cells (Genes Cells. 2005 June; 10(6):503-16.).

Gordon et al. demonstrated the induction of brachyury [positive]/HNF3beta [positive] endoderm cells from mouse embryonic stem cells in theabsence of serum and in the presence of activin along with an inhibitorof Wnt signaling (US 2006/0003446A1).

Gordon et al. (PNAS, Vol 103, page 16806, 2006) states “Wnt andTGF-beta/nodal/activin signaling simultaneously were required for thegeneration of the anterior primitive streak”.

However, the mouse model of embryonic stem cell development may notexactly mimic the developmental program in higher mammals, such as, forexample, humans.

Thomson et al. isolated embryonic stem cells from human blastocysts(Science 282:114, 1998). Concurrently, Gearhart and coworkers derivedhuman embryonic germ (hEG) cell lines from fetal gonadal tissue(Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). Unlikemouse embryonic stem cells, which can be prevented from differentiatingsimply by culturing with Leukemia Inhibitory Factor (LIF), humanembryonic stem cells must be maintained under very special conditions(U.S. Pat. No. 6,200,806; WO 99/20741; WO 01/51616).

D'Amour et al. describes the production of enriched cultures of humanembryonic stem cell-derived definitive endoderm in the presence of ahigh concentration of activin and low serum (Nature Biotechnology 2005).Transplanting these cells under the kidney capsule of mice resulted indifferentiation into more mature cells with characteristics of someendodermal organs. Human embryonic stem cell-derived definitive endodermcells can be further differentiated into PDX1 positive cells afteraddition of FGF-10 (US 2005/0266554A1).

D'Amour et al. (Nature Biotechnology 24, 1392-1401 (2006)) states: “Wehave developed a differentiation process that converts human embryonicstem (hES) cells to endocrine cells capable of synthesizing thepancreatic hormones insulin, glucagon, somatostatin, pancreaticpolypeptide and ghrelin. This process mimics in vivo pancreaticorganogenesis by directing cells through stages resembling definitiveendoderm, gut-tube endoderm, pancreatic endoderm and endocrine precursoren route to cells that express endocrine hormones”.

In another example, Fisk et al. reports a system for producingpancreatic islet cells from human embryonic stem cells(US2006/0040387A1). In this case, the differentiation pathway wasdivided into three stages. Human embryonic stem cells were firstdifferentiated to endoderm using a combination of sodium butyrate andactivin A. The cells were then cultured with TGF-β antagonists such asNoggin in combination with EGF or betacellulin to generate PDX1 positivecells. The terminal differentiation was induced by nicotinamide.

In one example, Benvenistry et al. states: “We conclude thatover-expression of PDX1 enhanced expression of pancreatic enrichedgenes, induction of insulin expression may require additional signalsthat are only present in vivo” (Benvenistry et al., Stem Cells 2006;24:1923-1930).

In another example, Grapin-Botton et al. states: “Early activation ofNgn3 almost exclusively induced glucagon+ cells while depleting the poolof pancreas progenitors. As from E11.5, PDX-1 progenitors becamecompetent to differentiate into insulin [positive] and PP [positive]cells” (Johansson K A et al., Developmental Cell 12, 457-465, March2007).

For example, Diez et al. states; “At 9 and 10 weeks, most of theglucagon positive cells co-expressed insulin, although distinctinsulin-only cells were clearly detectable at these stages. Cellsco-expressing insulin and glucagon were observed during the whole periodstudies (9 to 21 weeks) but they represent merely a small fraction ofthe total insulin and glucagon expressing cells.” (J Histochem Cytochem.2009 September; 57(9):811-24. 2009 Apr. 13.)

In one example, Chen et al. states ““(−)-indolactam V [(ILV)] activatesprotein kinase C signaling and directs the pancreatic specification ofhESCs that have already been committed to the endoderm lineage . . . ILVand retinoic acid function through a related mechanism . . . ILV shows astronger induction of PDX-1 expressing cells (percentage of cellsexpressing PDX-1) than does retinoic acid.” (Nature Chemical Biology 5,195-196 (April 2009) doi:10.1038/nchembio0409-195).

Lyttle et al. states: “NKX6-1 co-localised only with insulin cells,indicating that NKX6-1 is exclusively involved in human beta celldevelopment.” (Diabetologia 2008 July: 51(7):1169-80, 2008).

Therefore, there still remains a significant need to develop in vitromethods to generate a functional insulin expressing cell, that moreclosely resemble a β cell. The present invention takes an alternativeapproach to improve the efficiency of differentiating human embryonicstem cells toward insulin expressing cells, by generating a populationof cells expressing markers characteristic of the pancreatic endocrinelineage that co-express NKX6.1 and insulin and minimal amounts ofglucagon.

SUMMARY

In one embodiment, the present invention provides a population of cellsexpressing markers characteristic of the pancreatic endocrine lineagethat co-express NKX6.1 and insulin and minimal amounts of glucagon.

In one embodiment, the present invention provides a method todifferentiate a population of pluripotent stem cells into a populationof cells expressing markers characteristic of the pancreatic endocrinelineage that co-express NKX6.1 and insulin and minimal amounts ofglucagon, comprising the steps of:

-   -   a. Culturing the pluripotent stem cells,    -   b. Differentiating the pluripotent stem cells into cells        expressing markers characteristic of the definitive endoderm        lineage,    -   c. Differentiating the cells expressing markers characteristic        of the definitive endoderm lineage into cells expressing markers        characteristic of the pancreatic endoderm lineage, and    -   d. Differentiating the cells expressing markers characteristic        of the pancreatic endoderm lineage into cells expressing markers        characteristic of the pancreatic endocrine lineage that        co-express NKX6.1 and insulin and minimal amounts of glucagon,        by treating the cells expressing markers characteristic of the        pancreatic endoderm lineage with medium supplemented with a        protein kinase C activator.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show the effect of TPB treatment on the expression ofinsulin and glucagon in the cells of the present invention. Panels A andB show the expression of insulin and glucagon respectively, in cellstreated with TPB. Control populations of cells are shown in panels C andD.

FIGS. 2A-2D show the effect of various concentrations of TPB on theexpression of insulin and glucagon in cells treated according to themethods of the present invention. Panels A through D show the expressionof insulin and glucagon in populations of cells treated with TPB at thedoses indicated.

FIGS. 3A-3D show the effect of a protein kinase C inhibitor on theexpression of insulin and glucagon in cells treated according to themethods of the present invention. Panel A depicts the expression ofinsulin and glucagon in cells treated with TPB, and Panel C depicts thecorresponding DAPI staining). Panel B depicts the expression of insulinand glucagon in cells treated with TPB and GÖ 6976, and Panel D depictsthe corresponding DAPI staining).

FIGS. 4A-4C show the effect of various protein kinase C activators onthe expression of insulin in cells treated according to the methods ofthe present invention. Panel A shows the expression of insulin in cellstreated with TPB. Panel B shows the expression of insulin in cellstreated with ILV. Panel C shows the expression of insulin in cellstreated with PMA.

FIGS. 5A-5E shows the expression of markers characteristic of thepancreatic endocrine lineage in cells treated according to the methodsof the present invention. The panels depict the expression of insulinand NKX6.1 (panel A), insulin and PDX1 (panel B), insulin and NEUROD1(panel C), insulin and somatostatin (panel D), and insulin and ghrelin(panel E).

FIGS. 6A-6F shows the expression of insulin and glucagon in cellstreated according to the methods of the present invention. Panels A to Cshow insulin expression (panel A), glucagon expression (panel B) andDAPI staining (panel C) in cells treated with DMEM-High glucose+1%B27+50 ng/ml FGF7+0.25 μM Cyclopamine-KAAD+2 μM Retinoic acid (RA)+100ng/ml of Noggin+20 ng/ml Activin A+a p38 kinase inhibitor (disclosed inU.S. Pat. No. 6,214,830, at 2.5 μM) for four days (Stage 3, Treatment 8,Example 2). Panels D to F show insulin expression (panel d), glucagonexpression (panel E) and DAPI staining (panel F) in cells treated withDMEM-High glucose+1% B27+0.25 μM Cyclopamine-KAAD+2 μM Retinoic acid(RA)+100 ng/ml of Noggin for four days (Stage 3, Treatment 9, Example2).

FIG. 7 shows human C-peptide was detected in (SCID)—beige (Bg) micefour, eight and twelve weeks after receiving the cells of the presentinvention, following a glucose challenge.

FIG. 8 shows the percentage of cells co-expressing PDX1 and NKX6.1obtained following treatment of various protein kinase C inhibitors, atthe concentrations indicated.

FIG. 9 shows the expression of NGN3, PDX1, NKX6.1 and PTF1 alpha incells treated according to the methods described in Example 6.

DETAILED DESCRIPTION

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the following subsectionsthat describe or illustrate certain features, embodiments orapplications of the present invention.

Definitions

Stem cells are undifferentiated cells defined by their ability at thesingle cell level to both self-renew and differentiate to produceprogeny cells, including self-renewing progenitors, non-renewingprogenitors, and terminally differentiated cells. Stem cells are alsocharacterized by their ability to differentiate in vitro into functionalcells of various cell lineages from multiple germ layers (endoderm,mesoderm and ectoderm), as well as to give rise to tissues of multiplegerm layers following transplantation and to contribute substantially tomost, if not all, tissues following injection into blastocysts.

Stem cells are classified by their developmental potential as: (1)totipotent, meaning able to give rise to all embryonic andextraembryonic cell types; (2) pluripotent, meaning able to give rise toall embryonic cell types; (3) multipotent, meaning able to give rise toa subset of cell lineages but all within a particular tissue, organ, orphysiological system (for example, hematopoietic stem cells (HSC) canproduce progeny that include HSC (self-renewal), blood cell restrictedoligopotent progenitors, and all cell types and elements (e.g.,platelets) that are normal components of the blood); (4) oligopotent,meaning able to give rise to a more restricted subset of cell lineagesthan multipotent stem cells; and (5) unipotent, meaning able to giverise to a single cell lineage (e.g., spermatogenic stem cells).

Differentiation is the process by which an unspecialized (“uncommitted”)or less specialized cell acquires the features of a specialized cellsuch as, for example, a nerve cell or a muscle cell. A differentiated ordifferentiation-induced cell is one that has taken on a more specialized(“committed”) position within the lineage of a cell. The term“committed”, when applied to the process of differentiation, refers to acell that has proceeded in the differentiation pathway to a point where,under normal circumstances, it will continue to differentiate into aspecific cell type or subset of cell types, and cannot, under normalcircumstances, differentiate into a different cell type or revert to aless differentiated cell type. De-differentiation refers to the processby which a cell reverts to a less specialized (or committed) positionwithin the lineage of a cell. As used herein, the lineage of a celldefines the heredity of the cell, i.e., which cells it came from andwhat cells it can give rise to. The lineage of a cell places the cellwithin a hereditary scheme of development and differentiation. Alineage-specific marker refers to a characteristic specificallyassociated with the phenotype of cells of a lineage of interest and canbe used to assess the differentiation of an uncommitted cell to thelineage of interest.

“Cells expressing markers characteristic of the definitive endodermlineage”, or “Stage 1 cells”, or “Stage 1”, as used herein, refers tocells expressing at least one of the following markers: SOX17, GATA4,HNF3 beta, GSC, CER1, Nodal, FGF8, Brachyury, Mix-like homeobox protein,FGF4 CD48, eomesodermin (EOMES), DKK4, FGF17, GATA6, CXCR4, C-Kit, CD99,or OTX2. Cells expressing markers characteristic of the definitiveendoderm lineage include primitive streak precursor cells, primitivestreak cells, mesendoderm cells and definitive endoderm cells.

“Cells expressing markers characteristic of the pancreatic endodermlineage”, as used herein, refers to cells expressing at least one of thefollowing markers: PDX1, NKX6.1, HNF1 beta, PTF1 alpha, HNF6, HNF4alpha, SOX9, HB9 or PROX1. Cells expressing markers characteristic ofthe pancreatic endoderm lineage include pancreatic endoderm cells,primitive gut tube cells, and posterior foregut cells.

“Definitive endoderm”, as used herein, refers to cells which bear thecharacteristics of cells arising from the epiblast during gastrulationand which form the gastrointestinal tract and its derivatives.Definitive endoderm cells express the following markers: HNF3 beta,GATA4, SOX17, Cerberus, OTX2, goosecoid, C-Kit, CD99, and MIXL1.

“Markers”, as used herein, are nucleic acid or polypeptide moleculesthat are differentially expressed in a cell of interest. In thiscontext, differential expression means an increased level for a positivemarker and a decreased level for a negative marker. The detectable levelof the marker nucleic acid or polypeptide is sufficiently higher orlower in the cells of interest compared to other cells, such that thecell of interest can be identified and distinguished from other cellsusing any of a variety of methods known in the art.

“Pancreatic endocrine cell”, or “Pancreatic hormone expressing cell”, or“Cells expressing markers characteristic of the pancreatic endocrinelineage” as used herein, refers to a cell capable of expressing at leastone of the following hormones: insulin, glucagon, somatostatin, andpancreatic polypeptide.

Isolation, Expansion and Culture of Pluripotent Stem CellsCharacterization of Pluripotent Stem Cells

Pluripotent stem cells may express one or more of the stage-specificembryonic antigens (SSEA) 3 and 4, and markers detectable usingantibodies designated Tra-1-60 and Tra-1-81 (Thomson et al., Science282:1145, 1998). Differentiation of pluripotent stem cells in vitroresults in the loss of SSEA-4, Tra 1-60, and Tra 1-81 expression (ifpresent) and increased expression of SSEA-1. Undifferentiatedpluripotent stem cells typically have alkaline phosphatase activity,which can be detected by fixing the cells with 4% paraformaldehyde, andthen developing with Vector Red as a substrate, as described by themanufacturer (Vector Laboratories, Burlingame Calif.). Undifferentiatedpluripotent stem cells also typically express OCT4 and TERT, as detectedby RT-PCR.

Another desirable phenotype of propagated pluripotent stem cells is apotential to differentiate into cells of all three germinal layers:endoderm, mesoderm, and ectoderm tissues. Pluripotency of pluripotentstem cells can be confirmed, for example, by injecting cells into severecombined immunodeficient (SCID) mice, fixing the teratomas that formusing 4% paraformaldehyde, and then examining them histologically forevidence of cell types from the three germ layers. Alternatively,pluripotency may be determined by the creation of embryoid bodies andassessing the embryoid bodies for the presence of markers associatedwith the three germinal layers.

Propagated pluripotent stem cell lines may be karyotyped using astandard G-banding technique and compared to published karyotypes of thecorresponding primate species. It is desirable to obtain cells that havea “normal karyotype,” which means that the cells are euploid, whereinall human chromosomes are present and not noticeably altered.

Sources of Pluripotent Stem Cells

The types of pluripotent stem cells that may be used include establishedlines of pluripotent cells derived from tissue formed after gestation,including pre-embryonic tissue (such as, for example, a blastocyst),embryonic tissue, or fetal tissue taken any time during gestation,typically but not necessarily before approximately 10 to 12 weeksgestation. Non-limiting examples are established lines of humanembryonic stem cells or human embryonic germ cells, such as, for examplethe human embryonic stem cell lines H1, H7, and H9 (WiCell). Alsocontemplated is use of the compositions of this disclosure during theinitial establishment or stabilization of such cells, in which case thesource cells would be primary pluripotent cells taken directly from thesource tissues. Also suitable are cells taken from a pluripotent stemcell population already cultured in the absence of feeder cells. Alsosuitable are mutant human embryonic stem cell lines, such as, forexample, BG01v (BresaGen, Athens, Ga.).

In one embodiment, human embryonic stem cells are prepared as describedby Thomson et al. (U.S. Pat. No. 5,843,780; Science 282:1145, 1998;Curr. Top. Dev. Biol. 38:133 ff., 1998; Proc. Natl. Acad. Sci. U.S.A.92:7844, 1995).

Culture of Pluripotent Stem Cells

In one embodiment, pluripotent stem cells are typically cultured on alayer of feeder cells that support the pluripotent stem cells in variousways. Alternatively, pluripotent stem cells are cultured in a culturesystem that is essentially free of feeder cells, but nonethelesssupports proliferation of pluripotent stem cells without undergoingsubstantial differentiation. The growth of pluripotent stem cells infeeder-free culture without differentiation is supported using a mediumconditioned by culturing previously with another cell type.Alternatively, the growth of pluripotent stem cells in feeder-freeculture without differentiation is supported using a chemically definedmedium.

For example, Reubinoff et al. (Nature Biotechnology 18: 399-404 (2000))and Thompson et al. (Science 6 Nov. 1998: Vol. 282. no. 5391, pp.1145-1147) disclose the culture of pluripotent stem cell lines fromhuman blastocysts using a mouse embryonic fibroblast feeder cell layer.

Richards et al., (Stem Cells 21: 546-556, 2003) evaluated a panel of 11different human adult, fetal and neonatal feeder cell layers for theirability to support human pluripotent stem cell culture. Richards et al.,states: “human embryonic stem cell lines cultured on adult skinfibroblast feeders retain human embryonic stem cell morphology andremain pluripotent”.

US20020072117 discloses cell lines that produce media that support thegrowth of primate pluripotent stem cells in feeder-free culture. Thecell lines employed are mesenchymal and fibroblast-like cell linesobtained from embryonic tissue or differentiated from embryonic stemcells. US20020072117 also discloses the use of the cell lines as aprimary feeder cell layer.

In another example, Wang et al. (Stem Cells 23: 1221-1227, 2005)discloses methods for the long-term growth of human pluripotent stemcells on feeder cell layers derived from human embryonic stem cells.

In another example, Stojkovic et al. (Stem Cells 2005 23: 306-314, 2005)disclose a feeder cell system derived from the spontaneousdifferentiation of human embryonic stem cells.

In a further example, Miyamoto et al. (Stem Cells 22: 433-440, 2004)disclose a source of feeder cells obtained from human placenta.

Amit et al. (Biol. Reprod 68: 2150-2156, 2003) discloses a feeder celllayer derived from human foreskin.

In another example, Inzunza et al. (Stem Cells 23: 544-549, 2005)disclose a feeder cell layer from human postnatal foreskin fibroblasts.

U.S. Pat. No. 6,642,048 discloses media that support the growth ofprimate pluripotent stem (pPS) cells in feeder-free culture, and celllines useful for production of such media. U.S. Pat. No. 6,642,048states: “This invention includes mesenchymal and fibroblast-like celllines obtained from embryonic tissue or differentiated from embryonicstem cells. Methods for deriving such cell lines, processing media, andgrowing stem cells using the conditioned media are described andillustrated in this disclosure.”

In another example, WO2005014799 discloses conditioned medium for themaintenance, proliferation and differentiation of mammalian cells.WO2005014799 states: “The culture medium produced in accordance with thepresent invention is conditioned by the cell secretion activity ofmurine cells; in particular, those differentiated and immortalizedtransgenic hepatocytes, named MMH (Met Murine Hepatocyte).”

In another example, Xu et al. (Stem Cells 22: 972-980, 2004) disclosesconditioned medium obtained from human embryonic stem cell derivativesthat have been genetically modified to over express human telomerasereverse transcriptase.

In another example, US20070010011 discloses a chemically defined culturemedium for the maintenance of pluripotent stem cells.

An alternative culture system employs serum-free medium supplementedwith growth factors capable of promoting the proliferation of embryonicstem cells. For example, Cheon et al. (BioReprodDOI:10.1095/biolreprod.105.046870, Oct. 19, 2005) disclose afeeder-free, serum-free culture system in which embryonic stem cells aremaintained in unconditioned serum replacement (SR) medium supplementedwith different growth factors capable of triggering embryonic stem cellself-renewal.

In another example, Levenstein et al. (Stem Cells 24: 568-574, 2006)disclose methods for the long-term culture of human embryonic stem cellsin the absence of fibroblasts or conditioned medium, using mediasupplemented with bFGF.

In another example, US20050148070 discloses a method of culturing humanembryonic stem cells in defined media without serum and withoutfibroblast feeder cells, the method comprising: culturing the stem cellsin a culture medium containing albumin, amino acids, vitamins, minerals,at least one transferrin or transferrin substitute, at least one insulinor insulin substitute, the culture medium essentially free of mammalianfetal serum and containing at least about 100 ng/ml of a fibroblastgrowth factor capable of activating a fibroblast growth factor signalingreceptor, wherein the growth factor is supplied from a source other thanjust a fibroblast feeder layer, the medium supported the proliferationof stem cells in an undifferentiated state without feeder cells orconditioned medium.

In another example, US20050233446 discloses a defined media useful inculturing stem cells, including undifferentiated primate primordial stemcells. In solution, the media is substantially isotonic as compared tothe stem cells being cultured. In a given culture, the particular mediumcomprises a base medium and an amount of each of bFGF, insulin, andascorbic acid necessary to support substantially undifferentiated growthof the primordial stem cells.

In another example, U.S. Pat. No. 6,800,480 states “In one embodiment, acell culture medium for growing primate-derived primordial stem cells ina substantially undifferentiated state is provided which includes a lowosmotic pressure, low endotoxin basic medium that is effective tosupport the growth of primate-derived primordial stem cells. The basicmedium is combined with a nutrient serum effective to support the growthof primate-derived primordial stem cells and a substrate selected fromthe group consisting of feeder cells and an extracellular matrixcomponent derived from feeder cells. The medium further includesnon-essential amino acids, an anti-oxidant, and a first growth factorselected from the group consisting of nucleosides and a pyruvate salt.”

In another example, US20050244962 states: “In one aspect the inventionprovides a method of culturing primate embryonic stem cells. Onecultures the stem cells in a culture essentially free of mammalian fetalserum (preferably also essentially free of any animal serum) and in thepresence of fibroblast growth factor that is supplied from a sourceother than just a fibroblast feeder layer. In a preferred form, thefibroblast feeder layer, previously required to sustain a stem cellculture, is rendered unnecessary by the addition of sufficientfibroblast growth factor.”

In a further example, WO2005065354 discloses a defined, isotonic culturemedium that is essentially feeder-free and serum-free, comprising: a. abasal medium; b. an amount of bFGF sufficient to support growth ofsubstantially undifferentiated mammalian stem cells; c. an amount ofinsulin sufficient to support growth of substantially undifferentiatedmammalian stem cells; and d. an amount of ascorbic acid sufficient tosupport growth of substantially undifferentiated mammalian stem cells.

In another example, WO2005086845 discloses a method for maintenance ofan undifferentiated stem cell, said method comprising exposing a stemcell to a member of the transforming growth factor-beta (TGF-β) familyof proteins, a member of the fibroblast growth factor (FGF) family ofproteins, or nicotinamide (NIC) in an amount sufficient to maintain thecell in an undifferentiated state for a sufficient amount of time toachieve a desired result.

The pluripotent stem cells may be plated onto a suitable culturesubstrate. In one embodiment, the suitable culture substrate is anextracellular matrix component, such as, for example, those derived frombasement membrane or that may form part of adhesion moleculereceptor-ligand couplings. In one embodiment, a the suitable culturesubstrate is MATRIGEL® (Becton Dickenson). MATRIGEL® is a solublepreparation from Engelbreth-Holm Swarm tumor cells that gels at roomtemperature to form a reconstituted basement membrane.

Other extracellular matrix components and component mixtures aresuitable as an alternative. Depending on the cell type beingproliferated, this may include laminin, fibronectin, proteoglycan,entactin, heparan sulfate, and the like, alone or in variouscombinations.

The pluripotent stem cells may be plated onto the substrate in asuitable distribution and in the presence of a medium that promotes cellsurvival, propagation, and retention of the desirable characteristics.All these characteristics benefit from careful attention to the seedingdistribution and can readily be determined by one of skill in the art.

Suitable culture media may be made from the following components, suchas, for example, Dulbecco's modified Eagle's medium (DMEM), Gibco#11965-092; Knockout Dulbecco's modified Eagle's medium (KO DMEM), Gibco#10829-018; Ham's F12/50% DMEM basal medium; 200 mM L-glutamine, Gibco#15039-027; non-essential amino acid solution, Gibco 11140-050;β-mercaptoethanol, Sigma #M7522; human recombinant basic fibroblastgrowth factor (bFGF), Gibco #13256-029.

Formation of Cells Expressing Markers Characteristic of the PancreaticEndocrine Lineage from Pluripotent Stem Cells

In one embodiment, the present invention provides a method for producingcells expressing markers characteristic of the pancreatic endodermlineage from pluripotent stem cells, comprising the steps of:

-   -   a. Culturing pluripotent stem cells,    -   b. Differentiating the pluripotent stem cells into cells        expressing markers characteristic of the definitive endoderm        lineage,    -   c. Differentiating the cells expressing markers characteristic        of the definitive endoderm lineage into cells expressing markers        characteristic of the pancreatic endoderm lineage, and    -   d. Differentiating the cells expressing markers characteristic        of the pancreatic endoderm lineage into cells expressing markers        characteristic of the pancreatic endocrine lineage.

In one aspect of the present invention, the cells expressing markerscharacteristic of the pancreatic endocrine lineage co-express NKX6.1 andinsulin and minimal amounts of glucagon.

Differentiation of Pluripotent Stem Cells into Cells Expressing MarkersCharacteristic of the Definitive Endoderm Lineage

Formation of cells expressing markers characteristic of the definitiveendoderm lineage may be determined by testing for the presence of themarkers before and after following a particular protocol. Pluripotentstem cells typically minimal amounts of such markers. Thus,differentiation of pluripotent cells is detected when cells begin toexpress them.

Pluripotent stem cells may be differentiated into cells expressingmarkers characteristic of the definitive endoderm lineage by any methodin the art or by any method proposed in this invention.

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineageaccording to the methods disclosed in D'Amour et al., NatureBiotechnology 23, 1534-1541 (2005).

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineageaccording to the methods disclosed in Shinozaki et al., Development 131,1651-1662 (2004).

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineageaccording to the methods disclosed in McLean et al., Stem Cells 25,29-38 (2007).

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineageaccording to the methods disclosed in D'Amour et al., NatureBiotechnology 24, 1392-1401 (2006).

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineage byculturing the pluripotent stem cells in medium containing activin A inthe absence of serum, then culturing the cells with activin A and serum,and then culturing the cells with activin A and serum of a differentconcentration. An example of this method is disclosed in NatureBiotechnology 23, 1534-1541 (2005).

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineage byculturing the pluripotent stem cells in medium containing activin A inthe absence of serum, then culturing the cells with activin A with serumof another concentration. An example of this method is disclosed inD'Amour et al., Nature Biotechnology, 2005.

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineage byculturing the pluripotent stem cells in medium containing activin A anda Wnt ligand in the absence of serum, then removing the Wnt ligand andculturing the cells with activin A with serum. An example of this methodis disclosed in Nature Biotechnology 24, 1392-1401 (2006).

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineage bytreating the pluripotent stem cells according to the methods disclosedin U.S. patent application Ser. No. 11/736,908, assigned to LifeScan,Inc.

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineage bytreating the pluripotent stem cells according to the methods disclosedin U.S. patent application Ser. No. 11/779,311, assigned to LifeScan,Inc.

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineage bytreating the pluripotent stem cells according to the methods disclosedin U.S. patent application Ser. No. 60/990,529.

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineage bytreating the pluripotent stem cells according to the methods disclosedin U.S. patent application Ser. No. 61/076,889.

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineage bytreating the pluripotent stem cells according to the methods disclosedin U.S. patent application Ser. No. 61/076,900.

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineage bytreating the pluripotent stem cells according to the methods disclosedin U.S. patent application Ser. No. 61/076,908.

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineage bytreating the pluripotent stem cells according to the methods disclosedin U.S. patent application Ser. No. 61/076,915.

Differentiation of Cells Expressing Markers Characteristic of theDefinitive Endoderm Lineage into Cells Expressing Markers Characteristicof the Pancreatic Endoderm Lineage

Cells expressing markers characteristic of the definitive endodermlineage may be differentiated into cells expressing markerscharacteristic of the pancreatic endoderm lineage by any method in theart or by any method proposed in this invention.

For example, cells expressing markers characteristic of the definitiveendoderm lineage may be differentiated into cells expressing markerscharacteristic of the pancreatic endoderm lineage according to themethods disclosed in D'Amour et al., Nature Biotechnol. 24:1392-1401,2006.

In one embodiment, the cells expressing markers characteristic of thepancreatic endoderm lineage co-express PDX1, NKX6.1, but minimal amountsof CDX2 and NGN3.

In one embodiment, cells expressing markers characteristic of thedefinitive endoderm lineage are differentiated into cells expressingmarkers characteristic of the pancreatic endoderm lineage thatco-express PDX1, NKX6.1, but minimal amounts of CDX2 and NGN3, byculturing the cells expressing markers characteristic of the definitiveendoderm lineage in a first medium supplemented with FGF7, followed byculturing the cells in a second medium supplemented with FGF7, a factorcapable of inhibiting BMP, a TGFβ receptor agonist, retinoic acid, and ahedgehog signaling pathway inhibitor.

In one embodiment, FGF7 may be used at a concentration from about 50pg/ml to about 50 μg/ml. In one embodiment, FGF7 is used at aconcentration of 50 ng/ml.

In one embodiment, the factor capable of inhibiting BMP is noggin.Noggin may be used at a concentration from about 500 ng/ml to about 500μg/ml. In one embodiment, noggin is used at a concentration of 100ng/ml.

In one embodiment, the TGFβ receptor agonist is selected from the groupconsisting of activin A, activin B, GDF-8, and GDF-11.

Activin A may be used at a concentration from about 2 ng/ml to 100ng/ml. In one embodiment, activin A is used at a concentration of 20ng/ml. In an alternate embodiment, activin A is used at a concentrationof 50 ng/ml.

Activin B may be used at a concentration from about 2 ng/ml to 100ng/ml. In one embodiment, activin B is used at a concentration of 20ng/ml. In an alternate embodiment, activin B is used at a concentrationof 50 ng/ml.

TGFβ-I may be used at a concentration from about 2 ng/ml to 100 ng/ml.In one embodiment, TGFβ-I is used at a concentration of 20 ng/ml. In analternate embodiment, TGFβ-I is used at a concentration of 50 ng/ml.

TGFβ-II may be used at a concentration from about 2 ng/ml to 100 ng/ml.In one embodiment, TGFβ-II is used at a concentration of 20 ng/ml. In analternate embodiment, TGFβ-II is used at a concentration of 50 ng/ml.

GDF-8 may be used at a concentration from about 2 ng/ml to 100 ng/ml. Inone embodiment, GDF-8 is used at a concentration of 20 ng/ml. In analternate embodiment, GDF-8 is used at a concentration of 50 ng/ml.

GDF-11 may be used at a concentration from about 2 ng/ml to 100 ng/ml.In one embodiment, GDF-11 is used at a concentration of 20 ng/ml. In analternate embodiment, GDF-11 is used at a concentration of 50 ng/ml.

Retinoic acid may be used at a concentration from about 1 nM to about 1mM. In one embodiment, retinoic acid is used at a concentration of 1 μM.

In one embodiment, the hedgehog signaling pathway inhibitor iscyclopamine-KAAD.

Cyclopamine-KAAD may be used at a concentration from about 0.025 μM toabout 2.5 μM. In one embodiment, cyclopamine-KAAD is used at aconcentration of 0.25 μM.

The efficiency of differentiation may be determined by exposing atreated cell population to an agent (such as an antibody) thatspecifically recognizes a protein marker expressed by cells expressingmarkers characteristic of the definitive endoderm lineage.

Methods for assessing expression of protein and nucleic acid markers incultured or isolated cells are standard in the art. These includequantitative reverse transcriptase polymerase chain reaction (RT-PCR),Northern blots, in situ hybridization (see, e.g., Current Protocols inMolecular Biology (Ausubel et al., eds. 2001 supplement)), andimmunoassays such as immunohistochemical analysis of sectioned material,Western blotting, and for markers that are accessible in intact cells,flow cytometry analysis (FACS) (see, e.g., Harlow and Lane, UsingAntibodies: A Laboratory Manual, New York: Cold Spring Harbor LaboratoryPress (1998)).

Characteristics of pluripotent stem cells are well known to thoseskilled in the art, and additional characteristics of pluripotent stemcells continue to be identified. Pluripotent stem cell markers include,for example, the expression of one or more of the following: ABCG2,cripto, FOXD3, CONNEXIN43, CONNEXIN45, OCT4, SOX2, Nanog, hTERT, UTF1,ZFP42, SSEA-3, SSEA-4, Tra 1-60, Tra 1-81.

After treating pluripotent stem cells with the methods of the presentinvention, the differentiated cells may be purified by exposing atreated cell population to an agent (such as an antibody) thatspecifically recognizes a protein marker, such as CXCR4, expressed bycells expressing markers characteristic of the definitive endodermlineage.

Pluripotent stem cells suitable for use in the present inventioninclude, for example, the human embryonic stem cell line H9 (NIH code:WA09), the human embryonic stem cell line H1 (NIH code: WA01), the humanembryonic stem cell line H7 (NIH code: WA07), and the human embryonicstem cell line SA002 (Cellartis, Sweden). Also suitable for use in thepresent invention are cells that express at least one of the followingmarkers characteristic of pluripotent cells: ABCG2, cripto, CD9, FOXD3,CONNEXIN43, CONNEXIN45, OCT4, SOX2, Nanog, hTERT, UTF1, ZFP42, SSEA-3,SSEA-4, Tra 1-60, and Tra 1-81.

Markers characteristic of the definitive endoderm lineage are selectedfrom the group consisting of SOX17, GATA4, HNF3 beta, GSC, CER1, Nodal,FGF8, Brachyury, Mix-like homeobox protein, FGF4, CD48, eomesodermin(EOMES), DKK4, FGF17, GATA6, CXCR4, C-Kit, CD99, and OTX2. Suitable foruse in the present invention is a cell that expresses at least one ofthe markers characteristic of the definitive endoderm lineage. In oneaspect of the present invention, a cell expressing markerscharacteristic of the definitive endoderm lineage is a primitive streakprecursor cell. In an alternate aspect, a cell expressing markerscharacteristic of the definitive endoderm lineage is a mesendoderm cell.In an alternate aspect, a cell expressing markers characteristic of thedefinitive endoderm lineage is a definitive endoderm cell.

Markers characteristic of the pancreatic endoderm lineage are selectedfrom the group consisting of PDX1, NKX6.1, HNF1 beta, PTF1 alpha, HNF6,HNF4 alpha, SOX9, HB9 and PROX1. Suitable for use in the presentinvention is a cell that expresses at least one of the markerscharacteristic of the pancreatic endoderm lineage. In one aspect of thepresent invention, a cell expressing markers characteristic of thepancreatic endoderm lineage is a pancreatic endoderm cell.

Differentiation of Cells Expressing Markers Characteristic of thePancreatic Endoderm Lineage into Cells Expressing Markers of thePancreatic Endocrine Lineage

In one embodiment, cells expressing markers characteristic of thepancreatic endoderm lineage are further differentiated into cellsexpressing markers characteristic of the pancreatic endocrine lineage.

In one embodiment, the cells expressing markers characteristic of thepancreatic endoderm lineage co-express PDX1, NKX6.1, but minimal amountsof CDX2 and NGN3.

In one embodiment, the cells expressing markers characteristic of thepancreatic endocrine lineage co-express NKX6.1 and insulin and minimalamounts of glucagon.

In one embodiment, cells expressing markers characteristic of thepancreatic endoderm lineage are differentiated into cells expressingmarkers characteristic of the pancreatic endocrine lineage thatco-express NKX6.1 and insulin and minimal amounts of glucagon, byculturing the cells expressing markers characteristic of the pancreaticendoderm lineage in a medium supplemented with a factor capable ofinhibiting BMP, a TGFβ receptor signaling inhibitor, and a proteinkinase C activator.

In one embodiment, the factor capable of inhibiting BMP is noggin.Noggin may be used at a concentration from about 500 ng/ml to about 500μg/ml. In one embodiment, noggin is used at a concentration of 100ng/ml.

In one embodiment, the TGFβ receptor signaling inhibitor is an inhibitorof ALK5. In one embodiment, the inhibitor of ALK5 is ALK5 inhibitor II.The ALK5 inhibitor II may be used at a concentration from about 0.1 μMto about 10 μM. In one embodiment, ALK5 inhibitor II is used at aconcentration of 1 μM.

In one embodiment, the protein kinase C activator is selected from thegroup consisting of (2S,5S)-(E,E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4-pentadiemoylamino) benzolactam,Indolactam V, and phorbol-12-myristate-13-acetate. In one embodiment,the protein kinase C activator is (2S,5S)-(E,E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4-pentadiemoylamino) benzolactam.(2S,5S)-(E, E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4-pentadiemoylamino)benzolactam may be used at a concentration from about 20 nM to about 500nM. (2S,5S)-(E,E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4-pentadiemoylamino) benzolactam,Indolactam V, and phorbol-12-myristate-13-acetate is referred to hereinas “TPB”.

Markers characteristic of the pancreatic endocrine lineage are selectedfrom the group consisting of NEUROD, ISL1, PDX1, NKX6.1, NKX2.2, PAX4,and PAX6. In one embodiment, the cells expressing markers characteristicof the pancreatic endocrine lineage co-express NKX6.1 and insulin andminimal amounts of glucagon.

Therapies

In one aspect, the present invention provides a method for treating apatient suffering from, or at risk of developing, Type1 diabetes. In oneembodiment, the method involves culturing pluripotent stem cells,differentiating the pluripotent stem cells in vitro into cellsexpressing markers characteristic of the pancreatic endocrine lineage,and implanting the cells expressing markers characteristic of thepancreatic endocrine lineage into a patient.

In yet another aspect, this invention provides a method for treating apatient suffering from, or at risk of developing, Type 2 diabetes. Inone embodiment, the method involves culturing pluripotent stem cells,differentiating the pluripotent stem cells in vitro into cellsexpressing markers characteristic of the pancreatic endocrine lineage,and implanting the cells expressing markers characteristic of thepancreatic endocrine lineage into a patient.

If appropriate, the patient can be further treated with pharmaceuticalagents or bioactives that facilitate the survival and function of thetransplanted cells. These agents may include, for example, insulin,members of the TGF-β family, including TGF-β1, 2, and 3, bonemorphogenic proteins (BMP-2, -3, -4, -5, -6, -7, -11, -12, and -13),fibroblast growth factors-1 and -2, platelet-derived growth factor-AA,and -BB, platelet rich plasma, insulin growth factor (IGF-I, II) growthdifferentiation factor (GDF-5, -6, -7, -8, -10, -15), vascularendothelial cell-derived growth factor (VEGF), pleiotrophin, endothelin,among others. Other pharmaceutical compounds can include, for example,nicotinamide, glucagon like peptide-I (GLP-1) and II, GLP-1 and 2mimetibody,

Exendin-4, retinoic acid, parathyroid hormone, MAPK inhibitors, such as,for example, compounds disclosed in U.S. Published Application2004/0209901 and U.S. Published Application 2004/0132729.

The pluripotent stem cells may be differentiated into aninsulin-producing cell prior to transplantation into a recipient. In aspecific embodiment, the pluripotent stem cells are fully differentiatedinto β-cells, prior to transplantation into a recipient. Alternatively,the pluripotent stem cells may be transplanted into a recipient in anundifferentiated or partially differentiated state. Furtherdifferentiation may take place in the recipient.

Definitive endoderm cells or, alternatively, pancreatic endoderm cells,or, alternatively, β cells, may be implanted as dispersed cells orformed into clusters that may be infused into the hepatic portal vein.Alternatively, cells may be provided in biocompatible degradablepolymeric supports, porous non-degradable devices or encapsulated toprotect from host immune response. Cells may be implanted into anappropriate site in a recipient. The implantation sites include, forexample, the liver, natural pancreas, renal subcapsular space, omentum,peritoneum, subserosal space, intestine, stomach, or a subcutaneouspocket.

To enhance further differentiation, survival or activity of theimplanted cells, additional factors, such as growth factors,antioxidants or anti-inflammatory agents, can be administered before,simultaneously with, or after the administration of the cells. Incertain embodiments, growth factors are utilized to differentiate theadministered cells in vivo. These factors can be secreted by endogenouscells and exposed to the administered cells in situ. Implanted cells canbe induced to differentiate by any combination of endogenous andexogenously administered growth factors known in the art.

The amount of cells used in implantation depends on a number of variousfactors including the patient's condition and response to the therapy,and can be determined by one skilled in the art.

In one aspect, this invention provides a method for treating a patientsuffering from, or at risk of developing diabetes. This method involvesculturing pluripotent stem cells, differentiating the cultured cells invitro into a β-cell lineage, and incorporating the cells into athree-dimensional support. The cells can be maintained in vitro on thissupport prior to implantation into the patient. Alternatively, thesupport containing the cells can be directly implanted in the patientwithout additional in vitro culturing. The support can optionally beincorporated with at least one pharmaceutical agent that facilitates thesurvival and function of the transplanted cells.

Support materials suitable for use for purposes of the present inventioninclude tissue templates, conduits, barriers, and reservoirs useful fortissue repair. In particular, synthetic and natural materials in theform of foams, sponges, gels, hydrogels, textiles, and nonwovenstructures, which have been used in vitro and in vivo to reconstruct orregenerate biological tissue, as well as to deliver chemotactic agentsfor inducing tissue growth, are suitable for use in practicing themethods of the present invention. See, for example, the materialsdisclosed in U.S. Pat. Nos. 5,770,417, 6,022,743, 5,567,612, 5,759,830,6,626,950, 6,534,084, 6,306,424, 6,365,149, 6,599,323, 6,656,488, U.S.Published Application 2004/0062753 A1, U.S. Pat. Nos. 4,557,264 and6,333,029.

To form a support incorporated with a pharmaceutical agent, thepharmaceutical agent can be mixed with the polymer solution prior toforming the support. Alternatively, a pharmaceutical agent could becoated onto a fabricated support, preferably in the presence of apharmaceutical carrier. The pharmaceutical agent may be present as aliquid, a finely divided solid, or any other appropriate physical form.Alternatively, excipients may be added to the support to alter therelease rate of the pharmaceutical agent. In an alternate embodiment,the support is incorporated with at least one pharmaceutical compoundthat is an anti-inflammatory compound, such as, for example compoundsdisclosed in U.S. Pat. No. 6,509,369.

The support may be incorporated with at least one pharmaceuticalcompound that is an anti-apoptotic compound, such as, for example,compounds disclosed in U.S. Pat. No. 6,793,945.

The support may also be incorporated with at least one pharmaceuticalcompound that is an inhibitor of fibrosis, such as, for example,compounds disclosed in U.S. Pat. No. 6,331,298.

The support may also be incorporated with at least one pharmaceuticalcompound that is capable of enhancing angiogenesis, such as, forexample, compounds disclosed in U.S. Published Application 2004/0220393and U.S. Published Application 2004/0209901.

The support may also be incorporated with at least one pharmaceuticalcompound that is an immunosuppressive compound, such as, for example,compounds disclosed in U.S. Published Application 2004/0171623.

The support may also be incorporated with at least one pharmaceuticalcompound that is a growth factor, such as, for example, members of theTGF-β family, including TGF-β1, 2, and 3, bone morphogenic proteins(BMP-2, -3,-4, -5, -6, -7, -11, -12, and -13), fibroblast growthfactors-1 and -2, platelet-derived growth factor-AA, and -BB, plateletrich plasma, insulin growth factor (IGF-I, II) growth differentiationfactor (GDF-5, -6, -8, -10, -15), vascular endothelial cell-derivedgrowth factor (VEGF), pleiotrophin, endothelin, among others. Otherpharmaceutical compounds can include, for example, nicotinamide, hypoxiainducible factor 1-alpha, glucagon like peptide-I (GLP-1), GLP-1 andGLP-2 mimetibody, and II, Exendin-4, nodal, noggin, NGF, retinoic acid,parathyroid hormone, tenascin-C, tropoelastin, thrombin-derivedpeptides, cathelicidins, defensins, laminin, biological peptidescontaining cell- and heparin-binding domains of adhesive extracellularmatrix proteins such as fibronectin and vitronectin, MAPK inhibitors,such as, for example, compounds disclosed in U.S. Published Application2004/0209901 and U.S. Published Application 2004/0132729.

The incorporation of the cells of the present invention into a scaffoldcan be achieved by the simple depositing of cells onto the scaffold.Cells can enter into the scaffold by simple diffusion (J. Pediatr. Surg.23 (1 Pt 2): 3-9 (1988)). Several other approaches have been developedto enhance the efficiency of cell seeding. For example, spinner flaskshave been used in seeding of chondrocytes onto polyglycolic acidscaffolds (Biotechnol. Prog. 14(2): 193-202 (1998)). Another approachfor seeding cells is the use of centrifugation, which yields minimumstress to the seeded cells and enhances seeding efficiency. For example,Yang et al. developed a cell seeding method (J. Biomed. Mater. Res.55(3): 379-86 (2001)), referred to as Centrifugational CellImmobilization (CCI). The present invention is further illustrated, butnot limited by, the following examples.

EXAMPLES Example 1 Formation of a Population of Cells Expressing MarkersCharacteristic of the Pancreatic Endocrine Lineage that Co-ExpressInsulin and NKX6.1 and Minimal Amounts of Glucagon

Cells of the human embryonic stem cells line H1 were cultured onMATRIGEL® (1:30 dilution) (BD Biosciences; Cat #356231)-coated disheswith RPMI medium (Invitrogen; Cat#: 22400)+0.2% FBS+100 ng/ml activin A(PeproTech; Cat #120-14)+20 ng/ml WNT-3a (R&D Systems; Cat #1324-WN/CF)for one day followed by treatment with RPMI media+0.5% FBS+100 ng/mlactivin A for an additional two days (Stage 1), then,

-   -   DMEM/F12(Invitrogen; Cat #11330-032)+2% FBS+50 ng/ml        FGF7(PeproTech; Cat #100-19) for three days (Stage 2), then    -   DMEM-High glucose (Invitrogen; Cat #10569)+1% B27+50 ng/ml        FGF7+0.25 μM Cyclopamine-KAAD (Calbiochem; Cat #239804) +100        ng/ml Noggin (R&D Systems; Cat #3344-NG)for four days (Stage 3),        then    -   DMEM-High glucose+1% B27 (Invitrogen; Cat #0791)+100 ng/ml        Noggin+1 μM ALK5 inhibitor II (Axxora; Cat #ALX-270-445)+500 nM        TBP ((2S,5S)-(E,        E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4-pentadiemoylamino)        benzolactam) (Calbiochem; Cat #565740)for six days (Stage 4).

As a control, separate populations of cells were treated with DMEM-Highglucose+1% B27+100 ng/ml Noggin+1 μM ALK5 inhibitor II for six days(Stage 4, control group). As shown in FIGS. 1A-1D, TBP treatment atstage 4 resulted in an increase of insulin-expressing cells (FIG. 1panel A). It was noted that about 60% of these insulin expressing cellsare single endocrine hormone expressing cells, wherein the cellsexpressed insulin and did not express glucagon somatostatin and ghrelin(FIG. 1 panel A and B, FIG. 5 panel D and E). Glucagon-expressing cellswere also noted in the cultures that received TBP treatment. Most of theglucagon-expressing cells also co-expressed insulin (FIG. 1 panel A andB). For the control group, the majority of the cells co-expressedinsulin and glucagon (FIG. 1 panel C and D).

In a separate experiment, cells of the human embryonic stem cells lineH1 were cultured on MATRIGEL® (1:30 dilution) coated dishes with RPMImedium+0.2% FBS+100 ng/ml activin A+20 ng/ml WNT-3a for one day followedby treatment with RPMI media+0.5% FBS+100 ng/ml activin A for anadditional two days (Stage 1), then

-   -   a. DMEM/F12+2% FBS+50 ng/ml FGF7 for three days (Stage 2), then    -   b. DMEM-High glucose+1% B27+0.25 μM Cyclopamine-KAAD+2 μM        Retinoic acid (RA)+100 ng/ml of Noggin for four days (Stage 3),        then    -   c. Treatment 1: DMEM-High glucose+1% B27+100 ng/ml Noggin+1 μM        ALK5 inhibitor II+500 nM TBP for six days (Stage 4, Treatment        1), or    -   d. Treatment 2: DMEM-High glucose+1% B27+100 ng/ml Noggin+1 μM        ALK5 inhibitor II+100 nM TBP for six days (Stage 4, Treatment        2), or    -   e. Treatment 3: DMEM-High glucose+1% B27+100 ng/ml Noggin+1 μM        ALK5 inhibitor II+20 nM TBP for six days (Stage 4, Treatment 3),        or    -   f. Treatment 4: DMEM-High glucose+1% B27+100 ng/ml Noggin+1 μM        ALK5 inhibitor II for six days (Stage 4, Treatment 4).

Immunocytochemistry analysis was used to assess the effects of thedifferent concentrations of TPB on the formation of the cells of thepresent invention. Significant increases in the number of singleinsulin-expressing cells was observed in both 500 nM and 100 nM TPBtreatment groups (FIG. 2 panel A and B). FACS analysis confirmed thatboth treatments gave rise to 12% single insulin expressing cells invitro and 15% of that population also expressed NKX6.1 (Table1—NKX6.1/INS expressing cells were 2.4% of the total population). At 20nM TPB, similar to the control group, most cells co-expressed insulinand glucagon (FIG. 2 panel C and D).

TABLE 1 Expression of markers characteristic of the pancreatic endocrinelineage, shown as a percentage of the total cell population.Synaptophysin INS NKX6.1 NKX6.1/INS TPB (500 nM) 38.3% 9.4% 45.7% 2.4%TPB (100 nM) 47.6% 14.4% 34.8% 3.1%

In order to further confirm that the effect on endocrine hormoneexpressing cell formation was mediated by the activation of proteinkinase C, separate populations of cells of the human embryonic stemcells line H1 were cultured on MATRIGEL® (1:30 dilution) coated disheswith RPMI medium+0.2% FBS+100 ng/ml activin A+20 ng/ml WNT-3a for oneday followed by treatment with RPMI media+0.5% FBS+100 ng/ml activin Afor an additional two days (Stage 1), then

-   -   DMEM/F12+2% FBS+50 ng/ml FGF7 for three days (Stage 2), then    -   DMEM-High glucose+1% B27+0.25 μM Cyclopamine-KAAD+2 μM Retinoic        acid (RA)+100 ng/ml of Noggin for four days (Stage 3), then    -   Treatment 5: DMEM-High glucose+1% B27+100 ng/ml Noggin+1 μM ALK5        inhibitor II+500 nM TBP for six days (Stage 4, Treatment 5), or    -   Treatment 6: DMEM-High glucose+1% B27+100 ng/ml Noggin+1 μM ALK5        inhibitor II+500 nM TPB+5 μM GÖ 6976 for six days (Stage 4,        Treatment 6), or    -   Treatment 7: DMEM-High glucose+1% B27+100 ng/ml Noggin+1 μM ALK5        inhibitor II (Stage 4, Treatment 7), then    -   DMEM-High glucose+1% B27 for four days (Stage 5).

GÖ 6976 is known to selectively inhibit Ca²⁺-dependent protein kinase Cisoforms. A significant decrease in the number cells expressing markerscharacteristic of the pancreatic endocrine lineage, in culturesreceiving TPB alone (FIG. 3, panel A) and TPB and GÖ 6976 (FIG. 3, panelB). FACS analysis confirmed that TPB treatment (Treatment 6) gave riseto 30.6% synaptophysin, 12% single insulin and 4.6% glucagon expressingcells. On the other hand, TBP and GÖ 6976 treatment (Treatment 7) gaverise to 10.6% synaptophysin and no detectable level of single insulinexpressing cells (Table 2). There was no difference in the total numberof cells observed between Treatment 6 and Treatment 7. (See FIG. 3,panels C and D, showing DAPI staining, reflecting total cell number inTreatment 6 and Treatment 7). These results suggest that protein kinaseC signaling may be important for the formation of cells expressingmarkers characteristic of the pancreatic endocrine lineage.

Other protein kinase C activators were also tested. These wereIndolactam V (ILV) (Axxora; Cat #ALX-420-011-C300) andphorbol-12-myristate-13-acetate (PMA) (Calbiochem; Cat #524400).However, only TPB demonstrated the formation of single insulinexpressing cells (FIG. 4, panel A). Both ILV (FIG. 4, panel B) and PMA(FIG. 4, panel C) at 500 nM, gave rise to cells co-expressing insulinand glucagon after six days. FACS analysis confirmed that TPB treatmentgave rise to 12% single insulin expressing, and 4.6% glucagon expressingcells and 7.1% insulin and glucagon co-expressing cells. On the otherhand, ILV treatment gave rise to 3% single insulin expressing, and 12%glucagon cells and 12% insulin and glucagon co-expressing cells (Table2).

Immunocytochemistry analysis showed that in cultures treated with TPB,20% of the insulin expressing cells co-expressed NKX6.1 (FIG. 5 panel A)and PDX1 (FIG. 5 panel B). The majority of the insulin expressing cellsco-expressed NEUROD, an endocrine maker (FIG. 5 panel C). Very few ofthe insulin expressing cells co-expressed somatostatin or ghrelin (GHRL)(FIG. 5 panel D and E).

TABLE 2 Expression of markers characteristic of the pancreatic endocrinelineage, shown as a percentage of the total cell population. TPB ILVTPB + Go6976 Synaptophysin 30.6%  56.8% 10.6% INS  12%   3% — GCG 4.6%12.6%  3.1% INS/GCG 7.1% 12.9%   4%

Example 2 An Alternative Method for the Formation of a Population ofCells Expressing Markers Characteristic of the Pancreatic EndocrineLineage that Co-Express Insulin and NKX6.1 and Minimal Amounts ofGlucagon

In a separate experiment, cells of the human embryonic stem cells lineH1 were cultured on MATRIGEL® (1:30 dilution) coated dishes with RPMImedium+0.2% FBS+100 ng/ml activin A+20 ng/ml WNT-3a for one day followedby treatment with RPMI media+0.5%FBS+100 ng/ml activin A for anadditional two days (Stage 1), then

-   -   DMEM/F12+2% FBS+50 ng/ml FGF7 for three days (Stage 2), then    -   Treatment 8: DMEM-High glucose+1% B27+50 ng/ml FGF7+0.25 μM        Cyclopamine-KAAD+2 μM Retinoic acid (RA)+100 ng/ml of Noggin+20        ng/ml Activin A+a p38 kinase inhibitor (disclosed in U.S. Pat.        No. 6,214,830, at 2.5 μM) for four days (Stage 3, Treatment 8),        or    -   Treatment 9: DMEM-High glucose+1% B27+0.25 μM Cyclopamine-KAAD+2        μM Retinoic acid (RA)+100 ng/ml of Noggin for four days (Stage        3, Treatment 9), then    -   DMEM-High glucose+1% B27+100 ng/ml Noggin+1 μM ALK5 inhibitor        II+500 nM TPB for six days (Stage 4).

Stage 3, Treatment 8 resulted in the formation of a population of cellsexpressing markers characteristic of the pancreatic endoderm lineagethat co-expressed PDX1 and NKX6.1, but did not express CDX2 and NGN3. Onthe other hand, stage 3, treatment 9 resulted in the formation of apopulation of cells expressing markers characteristic of the pancreaticendoderm lineage that co-expressed PDX1, NKX6.1 and NGN3. The effects oftreatment with protein kinase C activator treatment on these cellpopulations were examined (Stage 4 above).

FACS analysis was performed to ascertain the percentage of insulinsingle positive cells, glucagon single positive cells, insulin/glucagondouble positive cells, cells expressing NKX6.1 positive cells,insulin/NKX6.1 positive cells, and synaptophysin positive cells (a panendocrine marker).

As shown in Table 3, the cell population formed with Treatment 8 gaverise to a larger percentage of endocrine cells, as denoted bysynaptophysin expression: 49.7% of the total cell population expressedsynaptophysin. 27.8% of the total population was insulin single positivecells.

On the other hand, the cell population formed with treatment 9 only gaverise to 25.7% synaptophysin expressing cells.7.6% of the totalpopulation was single insulin-expressing cells. No significantdifference of single glucagon expressing cells was observed in bothtreatments and the percentage of glucagon expressing cells wassignificantly lower than the insulin expressing cells.

A significant amount of insulin-expressing cells also co-expressedNKX6.1. In populations of cells that received treatment 8, 11% of thetotal population expressed insulin and NKX6.1. In populations of cellsthat received treatment 9, 2% of the total population expressed insulinand NKX6.1.

Immunofluorescent analysis confirmed the above (FIGS. 6A-6F). Treatment8, resulted in an increase of insulin expressing cells comparing toTreatment 9 (FIG. 6 panel A and D). Most glucagon expressing cells werepoly-hormonal cells (FIG. 6, panel A, B, D and E). These results suggestthat the population of cells generated by treatment 8 (cells expressingmakers characteristic of the pancreatic endoderm lineage thatco-expressed PDX1 and NKX6.1, but did not express CDX2 and NGN3) can bemore efficiently induced to become mature and functional insulinexpressing cells by the methods of the present invention.

TABLE 3 Expression of markers characteristic of the pancreatic endocrinelineage, shown as a percentage of the total cell population.Synaptophysin Insulin Glucagon Insulin/Glucagon NKX6.1 NKX6.1/Insulin T849.7% 27.8% 2.0% 16.4% 44.2% 11.0% T9 25.7% 7.6% 2.5% 4.9% 61.7% 2.0%

Example 3 An Alternative Method for the Formation of a Population ofCells Expressing Markers Characteristic of the Pancreatic EndocrineLineage that Co-Express Insulin and NKX6.1 and Minimal Amounts ofGlucagon

In another experiment, cells of the human embryonic stem cells line H1were cultured on MATRIGEL® (1:30 dilution) coated dishes with RPMImedium+0.2% FBS+100 ng/ml activin A+20 ng/ml WNT-3a for one day followedby treatment with RPMI media+0.5%FBS+100 ng/ml activin A for anadditional two days (Stage 1), then

-   -   DMEM/F12+2% FBS+50 ng/ml FGF7 for three days (Stage 2), then    -   DMEM-High glucose+1% B27+50 ng/ml FGF7+0.25 μM        Cyclopamine-KAAD+2 μM Retinoic acid (RA)+100 ng/ml of Noggin+20        ng/ml Activin A+a p38 kinase inhibitor (JNJ3026582, at 2.5 μM)        for four days (Stage 3), then    -   Treatment 10: DMEM-High glucose+1% B27+100 ng/ml Noggin+1 μM        ALK5 inhibitor II+500 nM TPB for six days (Stage 4, Treatment        10), or    -   Treatment 11: DMEM-High glucose+1% B27+100 ng/ml Noggin+1 μM        ALK5 inhibitor II+500 nM TPB for nine days (Stage 4, Treatment        11), or    -   Treatment 12: DMEM-High glucose+1% B27+100 ng/ml Noggin+1 μM        ALK5 inhibitor II+500 nM TPB for twelve days (Stage 4, Treatment        12)

As shown in Table 4, when the duration of the protein kinase C activatortreatment was extended to either nine days (Treatment 11) or twelve days(Treatment 12), no additional benefit was observed. Singleinsulin-expressing cells were 27.8% of the total population after sixdays treatment with Treatment 10. Conversely, insulin-expressing cellsdecreased to 10% after nine days treatment (Treatment 11), and declinedfurther to 4% after twelve days treatment (Treatment 12). In parallel,the total percentage of insulin and NKX6.1 co-expressing cell populationalso dropped significantly after extending the treatment. These resultssuggested that a six day treatment with Noggin, Alk5 inhibitor II and aprotein kinase C activator was sufficient enough to form the cells ofthe present invention.

TABLE 4 Expression of markers characteristic of the pancreatic endocrinelineage, shown as a percentage of the total cell population.Synaptophysin Insulin Glucagon Insulin/Glucagon NKX6.1 NKX6.1/Insulin6-day 49.7% 27.8% 2.0% 16.4% 44.2% 11.0% 9-day 43.5% 10.0% 6.6% 7.8%33.5% 1.0% 12-day  37.6% 4.4%  4% 6.3% 32.5% 1.0%

Example 4 Implantation of the Cells of the Present Invention into SevereCombined Immunodeficient (SCID)—Beige (Bg) Mice

Cells of the human embryonic stem cells line H1 were cultured onMATRIGEL® (1:30 dilution)-coated dishes with RPMI medium+0.2% FBS+100ng/ml activin A+20 ng/ml WNT-3a for one day followed by treatment withRPMI media+0.5% FBS+100 ng/ml activin A for an additional two days(Stage 1), then,

-   -   DMEM/F12+2% FBS+50 ng/ml FGF7 for three days (Stage 2), then    -   DMEM-High glucose+1% B27+50 ng/ml FGF7+0.25 μM        Cyclopamine-KAAD+100 ng/ml Noggin for four days (Stage 3), then    -   DMEM-High glucose+1% B27+100 ng/ml Noggin+1 μM ALK5 inhibitor        II+500 nM TBP for six days (Stage 4).

Cells at the end of stage four were mechanically scored using a 1-mlglass pipette and subsequently transferred to non-adherent plates forculture overnight. The resulting cell aggregates were collected, andaggregates containing 5 million cells were transplanted into the kidneycapsule of an immuno-compromised mice (SCID/Bg, animal Nos. 47. 48. 49,50 and 51). See FIG. 7.

After four weeks, functionality of the insulin-producing cells in thesegrafts was tested by injecting animals with glucose to induce insulinsecretion. The animals were fasted for about 15-20 hrs, after which ablood sample (pre-glucose) was withdrawn retro-orbitally. Each animalthen received an intraperitoneal injection dose of approximately 3g/kgof glucose in 30% dextrose solution, and blood was withdrawn at about 60minutes post glucose infusion. Circulating human C-peptide was detectedusing in mouse serum using an ultra-sensitive human specific C-peptideELISA plates (Cat No. 80-CPTHU-E01, Alpco Diagnostics, NH). Thedetection of human C-peptide indicates that insulin secretion is derivedfrom the grafted cells.

Human C-peptide was detected in animal serum as early as 4 weeks aftertransplantation and increased over time. The transplantation data issummarized in FIG. 7. At the end of one month, we were able to detectthe human C-peptide (less than 0.2 ng/ml) in response to glucoseadministration in 60% of the animals in the study group. Glucosestimulated serum level of human C-peptide increased 5 to 10 fold inthree out of the four mice after four weeks. At twelve weeks postimplantation, the average glucose-stimulated serum levels of humanc-peptide in transplanted mice were greater than 1 mg/ml (n=4).

Example 5 An Alternative Method for the Formation of a Population ofCells Expressing Markers Characteristic of the Pancreatic EndodermLineage that Co-Express PDX1 and NKX6.1

Briefly, cells of the human embryonic stem cell line H1 were cultured onMATRIGEL® (1:30 dilution) coated dishes and RPMI medium supplementedwith 0.2% FBS, 100 ng/ml activin A and 20 ng/ml WNT-3a for one day,followed by treatment with RPMI media supplemented with 0.5% FBS and 100ng/ml activin A, for an additional two days (Stage 1), then

-   -   DMEM/F12+2% FBS+50 ng/ml FGF7 for three days (Stage 2), then    -   DMEM-High glucose+1% B27+0.25 μM Cyclopamine-KAAD+2 μM Retinoic        acid (RA)+100 ng/ml of Noggin for four days (Stage 3), then    -   DMEM-High glucose+1% B27+100 ng/ml Noggin+1 μM ALK5 inhibitor        II+20 nM PMA, or 100 nM TPB, or 20 nM Phorbol-12,13-dibutyrate        (PDBu) (Calbiochem, cat #524390) for six days (Stage 4)

As a control, separate populations of cells were treated with DMEM Highglucose, supplemented with 1% B27, 100 ng/ml of Noggin and 1 μM ALK5inhibitor II for six days (stage 4).

Cultures were sampled in duplicate on stage 4 day 6, and imaging wasperformed using an IN Cell Analyzer 1000 (GE Healthcare). Images from100 fields per well were acquired to compensate for any cell loss duringthe bioassay and subsequent staining procedures. Measurements for totalcell number, total PDX1 expressing cells and total NKX6.1 expressingcell were obtained from each well using IN Cell Developer Toolbox 1.7(GE Healthcare) software. Averages and standard deviations werecalculated for each replicate data set. Total PDX1 and NKX6.1 proteinexpressing cells was reported as percentage of the total cellpopulation. As shown in FIG. 8, there was a dramatic increase ofNKX6.1/PDX1 expressing cell population in the protein kinase C activatortreated groups at a lower effective concentration (approximately 20 nM),compared to samples obtained from the control treatment. By day 6 ofStage 4, in populations of cells that received either protein kinase Cactivator or control treatment, 92%±4% of the population expressed PDX1.In the protein kinase C activator treated group, 75%±5% PDX1-expressingcells expressed NKX6.1. However, in populations only treated with Nogginand TGF beta receptor inhibitor (control), only 58%±5% of thePDX1-expressing cells expressed NKX6.1. In the presence of proteinkinase C activator, 20% NKX6.1-expressing cells were co-positive withproliferation marker, EdU (Click-iT® EdU Imaging Kit, Invitrogen, Cat#C10337).

This example demonstrates that a protein kinase C activator can be usedin combination with Noggin and TGF beta receptor inhibitor at arelatively low effective concentration (˜20 nM) to facilitate theup-regulation of Nkx6.1 expression, and increase the percentage of cellsexpressing PDX1 and NKX6.1.

Example 6 Treatment of Cells Expressing Markers Characteristic of thePancreatic Endoderm Linage with Protein Kinase C Activators

Briefly, cells of the human embryonic stem cell line H1 were cultured onMATRIGEL® (1:30 dilution) coated dishes and RPMI medium supplementedwith 0.2% FBS, 100 ng/ml activin A and 20 ng/ml WNT-3a for one day,followed by treatment with RPMI media supplemented with 0.5% FBS and 100ng/ml activin A, for an additional two days (Stage 1), then

-   -   DMEM/F12+2% FBS+50 ng/ml FGF7 for three days (Stage 2), then    -   T1: DMEM-High glucose+1% B27+0.25 μM Cyclopamine-KAAD+2 μM        Retinoic acid (RA)+100 ng/ml of Noggin+FGF10 50 ng/ml for four        days (Stage 3, T1) or,    -   T2: DMEM-High glucose+1% B27+0.25 μM Cyclopamine-KAAD+2 μM        Retinoic acid (RA)+100 ng/ml of Noggin+FGF10 50 ng/ml+100 nM TPB        for four days (Stage 3, T2), then    -   DMEM-High glucose+1% B27+100 ng/ml Noggin+1 μM ALK5 inhibitor II        for six days (Stage 4)

As shown in FIG. 9, a significant down-regulation of the pancreaticendoderm markers PDX1, NKX6.1 and PTF1 alpha was observed, in cellstreated with TPB (T2) compared to the control group (T1). NKX6.1 wasundetectable by immunohistochemistry. These data suggest that proteinkinase activator treatment at stage 3 did not facilitate the generationof PDX1/NKX6.1 co-expressing cells.

Publications cited throughout this document are hereby incorporated byreference in their entirety. Although the various aspects of theinvention have been illustrated above by reference to examples andpreferred embodiments, it will be appreciated that the scope of theinvention is defined not by the foregoing description but by thefollowing claims properly construed under principles of patent law.

What is claimed is:
 1. A method of producing a population of NKX6.1 andinsulin co-expressing human pancreatic endocrine cells comprisingculturing pancreatic endoderm cells in a medium supplemented withnoggin, an inhibitor of ALK5, and a protein kinase C activator, whereinless than 10% of the cells in the population express glucagon.
 2. Themethod of claim 1, wherein the method comprises culturing for about 6 toabout 12 days.
 3. The method of claim 1, wherein the inhibitor of ALK5is ALK5 inhibitor II.
 4. The method of claim 1, wherein the proteinkinase C activator is(2S,5S)-(E,E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam.
 5. The method of claim 1, wherein at least 30% of the cellsexpress NKX6.1.
 6. The method of claim 1, wherein at least 40% of thecells express NKX6.1.
 7. The method of claim 1, wherein at least 50% ofthe cells express NKX6.1.
 8. The method of claim 1, wherein at least 60%of the cells express NKX6.1.
 9. The method of claim 1, wherein at least5% of the cells express insulin.
 10. A method of producing humanpancreatic endocrine cells comprising culturing pancreatic endodermcells in a medium supplemented with noggin, an inhibitor of ALK5, and aprotein kinase C activator.
 11. The method of claim 10, wherein lessthan 10% of the pancreatic endocrine cells in the population expressglucagon.
 12. The method of claim 10, wherein the method comprisesculturing for about 6 to about 12 days.
 13. The method of claim 10,wherein the inhibitor of ALK5 is ALK5 inhibitor II.
 14. The method ofclaim 10, wherein the protein kinase C activator is (2S,5S)-(E,E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4-pentadienoylamino) benzolactam.15. The method of claim 10, wherein at least 30% of the pancreaticendocrine cells express NKX6.1.
 16. The method of claim 10, wherein atleast 40% of the pancreatic endocrine cells express NKX6.1.
 17. Themethod of claim 10, wherein at least 50% of the pancreatic endocrinecells express NKX6.1.
 18. The method of claim 10, wherein at least 60%of the pancreatic endocrine cells express NKX6.1.
 19. The method ofclaim 10, wherein at least 5% of the pancreatic endocrine cells expressinsulin.